Structural proteins of fish pancreatic disease virus and uses thereof

ABSTRACT

The present invention relates to the structural proteins of the causative agent of Pancreatic Disease in fish, nucleotide sequences encoding said proteins, vaccines comprising said proteins or nucleotide sequences and diagnostic kits comprising said proteins or nucleotide sequences.

[0001] The present invention relates to the structural proteins of thecausative agent of Pancreatic Disease in fish, nucleotide sequencesencoding said proteins, vaccines comprising said proteins or nucleotidesequences and diagnostic kits comprising said proteins or nucleotidesequences.

[0002] Pancreatic Disease (PD) is a serious disease that affects fish,in particular salmonid fish such as wild Atlantic salmon, rainbow troutand the like. The disease causes lesions in the pancreas, including lossof pancreatic exocrine tissue, and fibrosis, cardiac and skeletal musclemyopathies. Outbreaks of PD were first described in 1984 by Munro et al,in Helgoland Meeresuntersuchungen 37:571-586 (1984). PD typicallyaffects the fish post-molts during the first year after they aretransferred to sea sites and is reported to spread rapidly among farmfish held in sea cages. Clinical signs include lethargy with a tendencyto congregate in cage corners and to fail to maintain a horizontalposition, cessation of feeding (anorexia) and significant mortalities(Ferguson et al, J. Fish Disease 9:95-98, 1986). Murphy et al (in J.Fish Disease 15:401-408, 1992) confirmed these observations in a laterstudy, in which it was found that cardiac and skeletal myopathy isexacerbated in fish suffering from PD.

[0003] An outbreak of PD in a fish farm can cause growth to be reducedand up to 10 percent of surviving fish may prove to be runt. On Irishfish farms PD causes significant mortality rates of 10 to 60 percentamong the young fish during the first year after they are transferred tosea sites (McLoughlin, M., Fish Farmer page 19, March/April 1995). Theestimated cost to the Irish industry in terms of loss of production iscurrently thought to be around £25 million per year. Consequently, thereis a great need for a vaccine for the prevention and/or treatment of PDin fish.

[0004] EP-A-712926 describes the isolation of the causative agent of PDfrom tissues of PD affected fish arid the identification of the virus asa toga-like virus. To prevent PD infections in fish, the use ofattenuated or inactivated PD for vaccination of the fish is accordinglysuggested. A drawback in the production of inactivated vaccines from thePD virus described in EP-A-712926 is the slow growth of the virus, inparticular on cell cultures, which makes the manufacturing of saidvaccines a relatively inefficient process. A further drawback with theinactivated vaccines is the instability of the inactivated virus in thepresence of other inactivated pathogens resulting in potency loss. Fishvaccines are generally produced as multivalent vaccines, and significanthigher amounts of inactivated virus are required in the multivalentvaccine than would be necessary in a monovalent vaccine to compensatefor the loss of potency.

[0005] The present invention provides the means to produce alternativevaccines to prevent infection of fish with PD, in which the abovementioned difficulties are overcome. The present invention provides forthe nucleotide sequence of the 3′ part of the genomic RNA of a salmon PDvirus (SPDV). This sequence of 5179 nucleotides is depicted in SEQ ID NO1 and contains several open reading frames (ORF's): On the coding strandnucleotide 2 to 1186 codes for a non-structural protein, and anotheroverlapping ORF starting from nucleotide 997 to 5076 codes for thestructural proteins. This ORF was designated as p130. Othernon-determined ORF's were found on the coding strand (3447 to 3767 and4289 to 4612) and the non coding strand (1207 to 890, and 1232-837).

[0006] The ORF from nucleotide 2 to 1186 codes for the C-terminal partof a non-structural protein designated as NSP4; its deduced amino acidis depicted in SEQ ID NO 2.

[0007] ORF p130 comprises the nucleotide sequences that encode thestructural proteins of the PD virus. The structural proteins of the PDvirus consist of a basic capsid protein, three envelope proteinsdesignated as E1, E2 and E3, and a protein designated as the 6K protein.The amino acid sequence of the whole protein encoded by the p130 ORF isdepicted in SEQ ID NO 3. After processing, the p130 protein is splicedinto the capsid protein (aa 76-375 of p130), E3 (aa 358-428 of p130), E2(aa429-866 of p130), 6K (aa 867-898 of p130), and E1 (aa 899-1359 ofp130).

[0008] The nucleotide sequence encoding the capsid protein of the PDvirus is located at nucleotide 1222 to 2067 of SEQ ID NO 1. Thecorresponding amino acid sequence (total 282 amino acids) is depicted inSEQ ID NO 4.

[0009] The nucleotide sequence encoding the envelope proteins E3, E2 andE1 are located at nucleotide 2068-2280, 2281-3594 and 3691-5076respectively, of the nucleotide sequence depicted in SEQ ID NO 1. Thecorresponding amino acid sequences of the E3, E2 and E1 proteins aredepicted in SEQ ID No's 5, 6 and 8 respectively.

[0010] The nucleotide sequence encoding the 6K protein is located atnucleotide 3595 to 3690 of the nucleotide sequence depicted in SEQ ID NO1, and the corresponding amino acid sequence of the 6K protein isdepicted in SEQ ID NO 7. Further sequence analysis of the viral RNAextracted from PD infected pancreas tissue revealed the existence of alonger variant of the 6K protein having 68 amino acids in lengthcompared to the 6K protein of 32 amino acids depicted in SEQ ID NO 7.The nucleotide sequence (SEQ ID NO 14) encoding the longer variant of 6Kprotein is 204 nucleotides in length compared to the 96 nucleotides ofthe nucleotide sequence encoding the truncated 6K protein. Thenucleotide sequence encoding the long variant of 6K protein and thededuced amino acid sequence thereof are shown in FIG. 2 and SEQ ID NO 14and SEQ ID NO 15 respectively.

[0011] The cloning and characterisation of the nucleotide sequences ofthe present invention provides for the production of the structuralproteins of the PD virus using standard recombinant DNA technology(Sambrooke et al., Molecular Cloning: a Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, 1989). Cloning techniquesand subsequent protein expression using in vitro expression systems arewell known in the art. In this way, recombinant structural PDV proteinscan be obtained, that are substantially free from other PDV proteins.These isolated structural proteins can be used to manufacture subunitvaccines to protect against infection of PD in fish. Said subunitvaccines may be used as marker vaccine in fish to distinguishvaccination from field infections with PD. Alternatively the nucleotidesequences encoding the structural proteins of the PD virus can be usedto manufacture DNA vaccines or vector vaccines to protect againstinfection of fish with PD. The nucleotide sequences and recombinant PDproteins can furthermore be used for diagnostic purposes, for instanceto detect the presence of PD virus in the field or anti-PD antibodies infish. Additionally, the recombinant PD proteins of the present inventioncan be used to produce PD specific antibodies. These antibodies can alsobe used for diagnostic purposes such as the detection of PD virus infish or in the field.

[0012] Thus, in a first aspect the invention provides for a nucleotidesequence comprising the nucleotide sequence depicted in SEQ ID NO 1encoding the structural proteins and part of NSP4 of the PD virus,fragments of said nucleotide sequence and a nucleotide sequencecomprising the nucleotide sequence depicted in SEQ ID NO 14. Preferredfragments of the nucleotide sequences according to the invention arenucleotide fragments 1222-5076 (also referred to as p130 encoding thecapsid, E3, E2, 6K and E1 proteins), 2068-5076 (also referred to as p98encoding the E3, E2, 6K and E1 proteins), 2068-3594 (also referred to aspE2 encoding E3 and E2 proteins), 1222-2067 (capsid), 2068-2280 (E3),2281-3594 (E2), 3595-3690 (6K), and 3691-50 (E1). For the purpose ofthis invention the nucleotide sequences according to the presentinvention also encompass the nucleotide sequence depicted in SEQ ID NO 1and fragment sequences thereof (such as the p130 and p98 fragments)which at least comprise a nucleotide sequence encoding for a 6K protein,wherein the nucleotide sequence depicted by nucleotide 3595-3690 of SEQID NO 1 has been substituted with the nucleotide sequence depicted inSEQ ID NO 14. Also within the scope of this invention are nucleotidesequences comprising tandem arrays of the nucleotide sequence comprisingthe sequence depicted in SEQ ID NO 1 or SEQ ID NO 14 or fragmentsthereof. Nucleotide sequences that are complementary to the sequencedepicted in SEQ ID NO 1, SEQ ID NO 14, or parts thereof are also withinthe scope of the invention, as well as nucleotide sequence thathybridise with the sequence depicted in SEQ ID NO 1 or SEQ ID NO 14. Thehybridisation conditions for this purpose are stringent, preferablyhighly stringent. According to the present invention the term“stringent” means washing conditions of 1×SSC, 0.1% SDS at a temperatureof 65° C.; highly stringent conditions refer to a reduction in SSCtowards 0.3×SSC.

[0013] Nucleotide sequences that hybridise with the sequence shown inSEQ ID NO 1 or SEQ ID NO 14 are understood to be nucleotide sequencesthat have a sequence homology of at least 70%, preferably 80%, morepreferably 90% with the corresponding matching part of the sequencedepicted in SEQ ID NO 1 or SEQ ID NO 14. According to the presentinvention the sequence homology is determined by comparing thenucleotide sequence with the corresponding part of the sequence depictedin SEQ ID NO 1 or SEQ ID NO 14. The sequence homology between anucleotide and the sequence in SEQ ID NO 1 or SEQ ID NO 14 candetermined via common sequence analysis program such as BLASTN and thelike. The optimal match area is automatically determined by theseprograms. Homologous sequences can easily be isolated from closelyrelated PD virus strains with the sequence depicted in SEQ ID NO 1 orSEQ ID NO 14 or fragments of these sequences using routine cloning andhybridisation techniques. Sleeping Disease (SD) virus is closely relatedto PD virus and the nucleic acid sequences encoding the structuralcapsid, E3, E2, E1 and 6K proteins of SD virus have the necssarysequence homology with the nucleic acid sequences depicted in SEQ ID NO1 and 14. Thus these SD nucleic acid sequences are also within thepresent invention.

[0014] The nucleotide sequences of the invention can be used in thepreparation of a DNA vaccine to vaccinate fish against PD infection. DNAvaccination refers to the induction of an immune response to one or moreantigens that are expressed in vivo from a gene inserted in a DNAplasmid which has been inoculated directly into the vaccinated fish.Thus in a second aspect of the invention there is provided for a DNAvaccine comprising a pharmaceutical acceptable carrier and a DNA plasmidin which a nucleotide sequence encoding one or more PDV structuralproteins is operably linked to a transcriptional regulatory sequence.

[0015] Preferably the nucleotide sequence to be used in said DNA plasmidis a nucleotide sequence comprising the nucleotide sequence depicted inSEQ ID NO 1 or a nucleotide sequence comprising the nucleotide sequencedepicted in SEQ ID NO 14 or fragments of said nucleotide sequences.Preferred fragments of the nucleotide sequence depicted in SEQ ID NO 1or 14 are nucleotide fragments 1222-5076, 2068-5076, 2068-3594,1222-2067, 2068-2280, 2281-3594, 3595-3690 3691-5076 of the sequencedepicted in SEQ ID NO 1, and combinations thereof such as for examplefragment 1222-2067 with fragment 2281-3594. Also suitable for use insaid DNA plasmid are nucleotide sequences that are complementary to thesequence of SEQ ID NO 1 or SEQ ID NO 14 or nucleotide sequences of whichthe sequence homology with the sequence depicted in SEQ ID NO 1 or SEQID NO 14 is at least 70%, preferably 80%, more preferably 90%. Thesequence homology between the nucleotide sequences that are suitable foruse in the DNA plasmid is determined as described earlier.

[0016] DNA plasmids that are suitable for use in a DNA vaccine accordingto the invention are conventional cloning or expression plasmids forbacterial, eukaryotic and yeast host cells, many of which arecommercially available. Well known examples of such plasmids are pBR322and pcDNA3 (Invitrogen). The DNA plasmids according to the inventionshould be able to induce protein expression of the nucleotide sequences.The DNA plasmid can comprise one or more nucleotide sequences accordingto the invention. In addition, the DNA plasmid can comprise othernucleotide sequences such as the immune-stimulating oligonucleotideshaving unmethylated CpG dinucleotides, or nucleotide sequences that codefor other antigenic proteins or adjuvating cytokines.

[0017] Transcriptional regulatory sequences that are suitable for use ina DNA plasmid according to the invention comprise promoters such as the(human) cytomegalovirus immediate early promoter (Seed, B. et al.,Nature 329, 840-842, 1987; Fynan, E. F. et al., PNAS 90,11478-11482,1993; Ulmer, J. B. et al., Science 259, 1745-1748, 1993),Rous sarcoma virus LTR (RSV, Gorman, C. M. et al., PNAS 79, 6777-6781,1982; Fynan et al., supra; Ulmer et al., supra), the-MPSV LTR (Stacey etal., J. Virology 50, 725-732, 1984), SV40 immediate early promoter(Sprague J. et al., J. Virology 45, 773, 1983), the metallothioneinpromoter (Brinster, R. L. et al., Nature 296, 39-42, 1982), the majorlate promoter of Ad2, the β-actin promoter (Tang et al., Nature 356,152-154, 1992). The regulatory sequences may also include terminator andpolyadenylation sequences. Amongst the sequences that can be used arethe well known bovine growth hormone polyadenylation sequence, the SV40polyadenylation sequence, the human cytomegalovirus (hCMV) terminatorand polyadenylation sequences.

[0018] The DNA plasmid comprising a nucleotide sequence according to thepresent invention operably linked to a transcriptional regulatorysequence for use in the vaccine according to the invention can be nakedor can be packaged in a delivery system. Suitable delivery systems arelipid vesicles, Iscoms, dendromers, niosomes, polysaccharide matrices,and the like. Also very suitable as delivery system are attenuated livebacteria such as Salmonella.

[0019] The nucleotide sequences according to the invention canadditionally be used in the production of a vector vaccine to vaccinatefish against PD. A vector vaccine is understood to be a vaccine in whicha live, attenuated bacteria or virus has been modified so that itcontains one or more heterologous nucleotide sequences inserted is intoits genetic material. These so called vector bacteria or viruses arecapable of coexpressing the heterologous proteins encoded by theinserted nucleotides. Thus in a third aspect the invention provides fora vector vaccine comprising a live attenuated bacteria or virus whichhave been modified to comprise in their genetic material one or more ofthe nucleotide sequences of the present invention. Very suitable for useas a vaccine vector are for example vaccinia virus or Semliki forestvirus

[0020] The nucleotide sequences according to the invention can also beused for the recombinant production of structural PD proteins,substantially free from other PD proteins. Thus in a fourth aspect theinvention provides for the structural proteins from PD virus. Morespecifically the invention provides for a PD capsid protein, the PDenvelope proteins E1, E2, and E3, and the 6K protein. In particularthere is provided for a capsid protein having the amino acid sequencedepicted in SEQ ID NO 4 or a derivative thereof, an E3 protein havingthe amino acid sequence depicted in SEQ ID NO 5 or a derivative thereof,an E2 protein having the amino acid sequence depicted in SEQ ID NO 6 ora derivative thereof, an E1 protein having the amino acid sequencedepicted in SEQ ID NO 8 or a derivative thereof, and a 6K protein havingthe amino acid sequence depicted in SEQ ID NO 7, SEQ ID NO 15 or aderivative thereof.

[0021] Derivative proteins are understood to be proteins which havealterations in the amino acid sequence(s) of the present invention whichdo not affect the antigenic and/or immunogenic characteristics of theseproteins, that is these derivative proteins are still capable ofinducing the production of antibodies that recognise and (cross)-reactwith the PD virus and/or inducing an immune response in fish thatprotects against PD infection. Antigenic characteristics are understoodto be the ability to induce production of antibodies that recognise and(cross)-react with the PD virus. Immunogenic characteristics areunderstood to be the ability to induce an immune response in fish thatprotects against infection with PD. The alterations that can occur in asequence according to the present invention could for instance resultfrom conservative amino acid substitutions, deletions, insertions,inversions or additions of (an) amino acid(s) in the overall sequence.Amino acid substitutions that are expected not to alter theimmunological properties have been described. Amino acid replacementsbetween related amino acids or replacements which have occurredfrequently in evolution are, inter alia Ser/Ala, Ser/Gly, Asp/Gly,Asp/Asn, Ile/Val (see Dayhof, M. D., Atlas of protein sequence andstructure, Nat. Biomed. Res. Found., Washington D.C., 1978, vol. 5,suppl. 3). Based on this information Lipman and Pearson developed amethod for rapid and sensitive protein comparison (Science, 1985, vol.227, 1435-1441) and determining the functional similarity betweenproteins and peptides having sequence homology. The derivative proteinsaccording to the invention are still capable to induce the production ofantibodies that recognise and (cross)-react with the PD virus and/or toinduce an immune response in the fish that protects against PDinfection. The capsid, E1, E2, E3, and 6K proteins derived from SleepingDisease (SD) virus are such derivative proteins according to theinvention. These proteins have an amino acid sequence that is identicalor almost identical to those of the PD virus as depicted in SEQ ID NO 4to 8 or 15. These proteins are capable to raise antibodies thatrecognize and cross-react with PD virus as well as SD virus. Otherderivatives are protein fragments that are still capable to induce theproduction of antibodies that recognise and (cross)-react with the PDvirus and/or to induce an immune response in the fish.

[0022] The proteins according to the invention can be prepared viastandard recombinant protein expression techniques. For this purpose anucleotide sequence encoding on or more of the proteins according to theinvention or a multimere of said protein is inserted into an expressionvector. Preferably the nucleotide sequence is a nucleotide sequencecomprising the nucleotide sequence depicted in SEQ ID NO 1 or SEQ ID NO14 or one or more fragments of these sequences. Preferred fragments ofthe nucleotide sequences according to the invention are nucleotidefragments 1222-10 5076, 2068-5076, 2068-3594, 1222-2067, 2068-2280,2281-3594, 3595-3690 3691-5076 of the sequence depicted in SEQ ID NO 1,and combinations thereof such as for example fragment 1222-2067 withfragment 2281-3594. Further preferred fragments according to theinvention are fragments of the nucleotide sequence depicted in SEQ ID NO15 such as for example the nucleotide sequence depicted by nucleotides3595-3690 of SEQ ID NO 1. Also suitable are nucleotide sequences thatare complementary to the sequence of SEQ ID NO 1 or SEQ ID NO 14 ornucleotide sequences of which the sequence homology with the sequencedepicted in SEQ ID NO 1 or SEQ ID NO 14 is at least 70%, preferably 80%,more preferably 90%. The sequence homology between the nucleotidesequences that are suitable for use in the DNA plasmid is determined asdescribed earlier.

[0023] Suitable expression vectors are, amongst others, plasmids,cosmids, viruses and YAC's (Yeast Artificial Chromosomes) which comprisethe necessary control regions for replication and expression. Theexpression vector can be brought to expression in a host cell. Suitablehost cells are, for instance, bacteria, yeast cells and mammalian cells.Such expression techniques are well known in the art (Sambrooke et al.,Molecular Cloning: a Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, 1989).The expressed proteins can be isolatedand purified from the medium. Expression of the whole p130 ORF(nucleotide fragment 997 to 5076 of SEQ ID NO 1) might lead to theforming of virus-like particles due to the spontaneous assemblance ofthe structural proteins.

[0024] The invention furthermore provides for a vaccine comprising oneor more of the structural PD proteins and a pharmaceutical acceptablecarrier. More specifically, a vaccine according to the inventioncomprises a capsid protein having an amino acid sequence depicted in SEQID NO 4 or a derivative thereof, an E3 protein having an amino acidsequence depicted in SEQ ID NO 5 or a derivative thereof, an E2 proteinhaving an amino acid sequence depicted in SEQ ID NO 6 or a derivativethereof, an E1 protein having an amino acid sequence depicted in SEQ IDNO 8 or a derivative thereof, a 6K protein having an amino acid sequencedepicted in SEQ ID NO 7 or SEQ ID NO 15 or a derivative thereof, or amixture comprising two or more of the proteins according to theinvention. Preferably the vaccine according to the invention comprisesthe E2 protein, and optionally the capsid protein. Also preferred is avaccine comprising all structural proteins of PD; these proteins canspontaneously form virus-like particles, thus providing a vaccine thatclosely resembles that of the whole pathogen. Vaccines according to theinvention are suitable for use as a marker vaccine to distinguishbetween vaccination and infection by PD in the field. A preferredvaccine according to the invention is a marker vaccine comprising a 6Kprotein having the amino acid sequence depicted in SEQ ID NO 7.

[0025] A vaccine according to the invention can be prepared according totechniques well known to the skilled practitioner. General techniquesfor the preparation of DNA vaccines have been widely described, forexample in EP patent 0 773 295 and U.S. Pat. No. 5,580,859.

[0026] Vaccines according to the invention comprise an effective amountof the afore-mentioned DNA plasmids, vector bacteria or virus, orproteins and a pharmaceutical acceptable carrier. The term “effective”as used herein is defined as the amount sufficient to induce an immuneresponse in the target fish. The amount of plasmid, vector or proteinwill depend on the type of plasmid or vector, the route ofadministration, the time of administration, the species of the fish aswell as age, general health and diet.

[0027] In general, a dosage of 0.01 to 1000 μg protein per kg bodyweight, preferably 0.5 to 500, more preferably 0.1 to 100 μg protein canbe used. With respect to the DNA vaccines, generally a minimum dosage of10 pg. up to dosages of 1000 μg of plasmid have been described to besufficient for a suitable expression of the antigens in vivo.

[0028] Pharmaceutical acceptable carriers that are suitable for use in avaccine according to the invention are sterile water, saline, aqueousbuffers such as PBS and the like. In addition a vaccine according to theinvention may comprise other additives such as adjuvants, stabilisers,anti-oxidants and others.

[0029] Suitable adjuvants include, amongst others, aluminium hydroxide,aluminium phosphate, amphigen, tocophenols, monophosphenyl lipid A,muramyl dipeptide, oil emulsions, glucans, carbomers, block-copolymers,cytokines and saponins such as Quil A. The amount of adjuvant addeddepends on the nature of the adjuvant itself.

[0030] Suitable stabilisers for use in a vaccine according to theinvention are for example carbohydrates including sorbitol, mannitol,starch, sucrose, dextrin, and glucose, proteins such as albumin orcasein, and buffers like alkaline phosphates.

[0031] The vaccines according to the invention are administered to thefish via injection, spray, immersion or peroral. The administrationprotocol can be optimised in accordance with standard vaccinationpractice.

[0032] The nucleotide sequences and the proteins according to theinvention are also suitable for use in diagnostics. The nucleotidesequences or fragments thereof can be used to detect the presence of PDvirus in the fish. A primer spanning the C-terminal part ofE2/6K/N-terminal part of E1 (see FIG. 2) was used in RT-PCR tosuccesfully detect the presence of PD virus in a clinical specimen of aPD outbreak. The proteins can be used to detect the presence ofantibodies in the fish.

[0033] The proteins according to the invention can additionally be usedfor the production of antibodies, using the general techniques availableto the practitioner in the field. Preferably the proteins are used toproduce specific monoclonal antibodies. The obtained antibodies may beutilised in diagnostics, to detect PD virus in the field, or in thefish.

[0034] Thus, in another aspect, the present invention provides for adiagnostic kit comprising one or more nucleotide sequences according tothe invention, or one or more structural proteins according to theinvention, or antibodies obtained with said proteins. Antibodiesaccording to the invention can be prepared according to standardtechniques. Procedures for immunising animals, e.g. mice with proteinsand selection of hybridomas producing immunogen specific monoclonalantibodies are well known in the art (see for example Coligan et al.(eds), Current protocols in Immunology, 1992; Kohler and Milstein,Nature 256:495-497, 1975; Steenbakkers et al., Mol. Biol. Rep.19:125-134, 1994).

[0035] The following examples are to illustrate the invention and shouldnot be interpreted to limit the invention in any way.

LEGENDS

[0036]FIG. 1: structural organisation of the various cloned nucleotidesequences coding for the PD structural proteins.

[0037]FIG. 2: Nucleotide sequence of C-terminus of E2 gene/“long”6Kgene/N-terminus of E1 gene. The putative cleavage sites between theE2/6K protein and 6K/E1 protein are represented by the vertical line (|)The nucleotide sequence encoding the “long”6K protein is 204 nucleotideslong and encodes a protein of 68 amino acids. The numbering betweenbrackets on the right of the sequence refers to the nucleotide- andamino acid residues of the 6K gene or protein respectively. Atnucleotide position. 44 of the nucleotide sequence encoding the 6K genethe G-residue can be replaced with an A residue, resulting in a 6Kprotein with an N residue at amino acid position 15 of the amino acidsequence depicted in the figure.

EXAMPLES

[0038] Cells and Virus

[0039] Isolation and cultivation of a salmon PD virus (SPDV) strain wascarried out in general as described in EP-A-712926. The F93125 isolateof SPDV was grown in Chinook salmon embryo (CHSE-214) cells aspreviously described (R. T. Nelson et al. (1995) Isolation of toga-likevirus from farmed Atlantic salmon Salmo salar with pancreas disease.Diseases of Aquatic Organisms 22, pp. 25-32). For virus purificationpurposes, monolayer cultures of CHSE-214 grown to ˜80% confluence in75cm² flasks were infected with I ml virus to give a multiplicity ofinfection of ˜1. After 1hr.adsorption an additional 14ml supplementedEagle's minimal essential medium (MEM) was introduced to each flask. Thevirus infected flasks were incubated at 15° C. for 7 or 8 days, whenvirus-induced cytopathic effect was evident, and the supernatant wascollected.

[0040] Virus Purification

[0041] The supernatant (typically 500 ml from virus-infected cells wasclarified at 3000 g for 20 min. Polyethyleneglycol (PEG) and NaCl wereadded to give final concentrations of 6% and 2.2% respectively.Following overnight incubation at 4° C. the PEG precipitate wascollected by centrifugation for 2 h at 3000 g. The resultant pellet wasresuspended in PBS (1-2 ml) and, after clarification at 1000 g for 5minutes, the crude virus suspension was fractionated by equilibriumdensity centrifugation using 11 ml gradients (20-60% w/w in PBS) ofsucrose. After centrifugation for 18 hr at 75000 g at 4° C., 1 mlfractions were collected from the bottom of the gradient. Fractionscontaining virus were identified by immunoblotting using an PD-specificmouse monoclonal antibody (Welsh et al., submitted 1999).

[0042] Production of PD Virus cDNA Clones

[0043] Viral RNA was extracted from gradient-purified PD virus andvirus-infected cells using RNA isolator (Genosys) and stored as ethanolprecipitates. A cDNA library was made by random priming with RNAextracted from gradient-purified virus. This library consisted of clonescontaining inserts (250-500 bp) in the vector pUC18 (Sureclone ligationKit, Pharmacia). Clones were selected randomly from the library andfollowing sequencing and analysis using the BLAST program (University ofWisconsin, Genetics Computer Group) were mapped to the alphavirusgenome. The sequences of three clones, N11, N38 and N50, were used todesign oligonucleotide primers that were used in reversetranscription-polymerase chain reaction (RT-PCR) to amplify 3overlapping fragments encompassing the 5.2 kb region at the 3′terminusof the PD genome. The incorporation of Not I sites into the primersfacilitated the restriction ligation of two of these fragments into theNot I site of vector pBluescript (Stratagene). PCR was carried out usingExpand Long Template PCR System (Boehringer Mannheim) at 94° C. for 30 s60° C. for 30 s, 68° C. for 2 min. Another clone was produced using3′RACE (M. A. Frohmann et al., 1998; Rapid production of full-lengthcDNA's from rare transcripts using a single gene-specificoligonucleotide primer. Proc. Natl. Acad. Sci.USA. 85, pp. 8998-9002).The reaction was performed using a 5′/3′ RACE kit (Boehringer Mannheim)with some modifications. Thus, RNA from gradient-purified virus wasindependently subjected to first-strand synthesis and the resultantcDNA's were amplified by PCR at 94° C. for 30 s, 60° C. for 30 s, 68° C.for I min.

[0044] Sequencing of PD Virus cDNA Clones

[0045] Cycle sequencing was performed using the ABI PRISM dye terminatorready reaction kit on purified plasmid DNA following the manufacturersprotocol (Perkin Elmer Cetus). Electropherograms were interpreted usingthe Sequence Navigator software (Perkin Elmer Cetus). The completenucleotide sequence of the 3′terminal 5.2 kb region of the PD virus RNAis presented in SEQ ID NO1.

[0046] An RT-PCR and sequence analysis using primers flanking theC-terminus of E2 and the N-terminus of E1 for viral RNA extracteddirectly from PD infected pancreas tissue revealed a longer 6K-encodingnucleotide sequence than the one depicted by nucleotides 3595-3690 ofSEQ ID NO 1. The nucleic acid encoding the full-length 6K protein aswell as the deduced amino acid sequence are shown in FIG. 2.

[0047] SPDV pFastBac1 and pcDNA3.1 (+) Constructs

[0048] Using standard cloning techniques (Sambrooke et al., MolecularCloning: a Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, 1989) four clones representing the SPDV structural regionhave been created in the vector pFastBac1 (Gibco BRL) for expression inthe baculovirus system. These clones have also been created in theexpression vector pcDNA3.1 (Invitrogen) for monoclonal antibodycharacterisation and use as a DNA vaccine. Details of how these cloneshave been produced are as follows:

[0049] Clone 1.

[0050] p130 encodes the complete structural gene region from the 1st ATGof the capsid protein to the poly(A) tract (3944nt). cDNA was producedfrom viral RNA by RT-PCR using the following primers: 5′ forward primer(5′130Not1): 5′-TGC ATG CGG CCG CAT GTT (SEQ ID NO 9) TCC CAT GCA ATTCAC CAA C-3′ 3′ inverse primer (3′130Not1) (sequence 5′ to 3′): 5′-TGCATG CGG CCG CTT GTA (SEQ ID NO 10) TTG AAA ATT TTA AAA CCA A-3′

[0051] These primers contain a 5 nucleotide stretch (ensures restrictionenzyme recognition) followed by a Not1 site then the appropriate SPDVsequence (highlighted in the attached sequence, from 1222 to 1245 for5′130Not1 and from 5143 to 5166 for 3′130Not1). The 3944nt cDNA productwas cloned into the Not1 site in both pFastBac1 and pcDNA3.1.

[0052] Clone 2.

[0053] p98 encodes for E3, E2, 6K and E1 to the poly(A) tract (3098nt).cDNA was produced from viral RNA by RT-PCR using the following primers:5′ forward primer (5′E3Not1): 5′-TGC ATG CGG CCG CAT GAC ACG (SEQ ID NO11) CGC TCC GGC CCT CCT G-3′ 3′ inverse primer (3′130Not1): 5′-TGC ATGCGG CCG CTT GTA TTG (SEQ ID NO 10) AAA ATT TTA AAA CCA A-3′

[0054] The primer 5′E3Not1 contains a 5 nucleotide stretch (ensuresrestriction enzyme recognition) followed by a Not1 site, an ATG(artificial start codon) then the appropriate SPDV sequence (from 2067to 2088) The primer 3′130Not1 is as described above in Clone1. The3098nt cDNA product was cloned into the Not1 site in both pFastBac1 andpcDNA3.1.

[0055] Clone 3.

[0056] pE2 encoding the E3 and E2 glycoproteins (1527nt). cDNA wasproduced from viral RNA by RT-PCR using the following primers: 5′forward primer (5′E3Not1): 5′-TGC ATG CGG CCG CAT GAC ACG (SEQ ID NO 11)CGC TCC GGC CCT CCT G-3′ 3′ inverse primer (3′E2Not1): 5′-TGC ATG CGGCCG CTC ACG CGC (SEQ ID NO 12) GAG CCC CTG GTA TGC AAC A-3′

[0057] The primer 5′E3Not1 is as described above in Clone2. The primer3′E2Not1 contains a 5 nucleotide stretch (ensures restriction enzymerecognition) followed by a Not1 site, a TGA (artificial stop codon) thenthe appropriate SPDV sequence (highlighted in the attached sequence,from 3571 to 3594). The 1527nt cDNA product was cloned into the Not1site in both pFastBac1 and pcDNA3.1.

[0058] Clone 4.

[0059] E2 encoding the E2 glycoprotein (1314nt). cDNA was produced fromviral RNA by RT-PCR using the following primers: 5′ forward primer(5′E2Not1): 5′-TGC ATG CGG CCG CAT GGC TGT (SEQ ID NO 13) GTC TAC GTCGCC TGC C-3′ 3′ inverse primer (3′E2Not1): 5′-TGC ATG CGG CCG CTC ACGCGC (SEQ ID NO 12) GAG CCC CTG GTA TGC AAC A-3′.

[0060] The primer 5′E2Not1 contains a 5 nucleotide stretch (ensuresrestriction enzyme recognition) followed by a Not1 site, an ATG(artificial start codon) then the appropriate SPDV sequence (from 2281to 2301). The primer 3′E2Not1 is as described above in Clone 3. The1314nt cDNA product was cloned into the Not1 site in both pFastBac1 andpcDNA3.1.

[0061] Insect cells (SF-9)were infected with the four recombinantbaculovirus constructs. Using monoclonals that were raised againstwhole-inactivated PD virus, an IFT staining was performed on theserecombinant baculovirus infected SF-9 cells. All produced proteinsreacted positively with the monoclonals, indicating that the recombinantproteins possess the wild-type epitopes.

[0062] Challenge Experiments

[0063] The proteins produced by all four constructs were collected usingTriton extraction. The proteins were BPL inactivated to prevent possiblespread of surviving recombinant baculoviruses in the environment. Theproteins were formulated into water-in-oil based vaccine formulationsand injected in a 0.2 ml vaccine volume

[0064] ELISA analysis using anti-PD-E2 monoclonals (2D9 capture and 7A2)showed that the amount of reactive epitopes per dose recombinant vaccinewas comparable or even higher than the amount of epitopes found in adose of the conventional inactivated PD virus vaccine.

[0065] A standardised challenge experiment performed at 8 weekspost-vaccination in Atlantic salmon fish showed that protection againstchallenge with salmon PD virus could be obtained with these recombinantsub-unit vaccines. In the experiment, lesions in pancreas, skeletalmuscle and heart muscle were scored in ordinal way. Significant levelswere calculated from Kruskal-Wallis one-way analysis of variance(non-parametric test). The vaccine formulation comprising the E2 orE2-E3 proteins gave similar levels of protection as obtained by theinactivated PD virus vaccine, while vaccines containing the recombinantproteins resulting from the p130 and p98 constructs respectively wereless protective then the inactivate PD virus vaccine.

[0066] Production of Antibodies.

[0067] DNA vaccination with proteins obtained from expression of thep130 nucleotide construct was carried out in mice to test for theantigenic properties of the recombinant proteins. After twoimntramuscular inoculations with p130-pcDNA3.1 recombinant expressionplasmids (see clone 1), the sera of mice showed an antibody reactionwith in vitro produced PD virus.

1 15 1 5179 DNA Salmon pancreatic disease virus 1 gactatggac tcagcggcaatgaacgtgga ggcttttaaa agtttcgcct gtaaggacac 60 cgacctgtgg actgagttcgcggaaaaacc agtaaggttg tcgcccggcc aaatcgaaga 120 gtatgtcttt catctacaaggggccaaggc caatgtgatg cacagcagag tcgaagccgt 180 atgccctgac ctctcggaggtggctatgga caggttcaca ctagacatga aacgcgacgt 240 caaagtgacg ccaggcacgaagcacgtaga ggagagacct aaagtccaag agattcaagc 300 ggccgacccc atggccaccgcgtacttgtg cgccatccat agagagctag tccgaaggct 360 gaaggccgtc ctgaaaccgtctatacacgt gttgttcgat atgagctccg aggattttga 420 tgctatcgtg ggccatgggatgaagttggg tgacaaggtg ctggaaacgg acatctcctc 480 attcgacaag agccaggaccaagccatggc ggttacagcg ctgatgctgc tgagggactt 540 gggagtagaa gaagacctcctgaccctaat tgaggcgtct ttcggcgaca tcacttctgc 600 ccacctgccc acaggcaccagatttcagtt tggatcgatg atgaagtctg gactttttct 660 gacgctgttc gtgaacacgctgcttaacat caccatagct gcccgagttt tacgggagca 720 gctggctgat accaggtgtgccgcgtttat cggtgacgac aacgtaatca ccggagtagt 780 gtctgacgac atgatggtggccaggtgcgc atcctggctg aacatggagg tgaagatcat 840 ggacatggaa attggcaacatgagtcctta tttttgtggc ggcttcctgt tactcgacac 900 ggtaacaggc actgtaagccgagtgtcgga ccctgtaaaa cgcctgatga agatgggaaa 960 accggccctg aacgatccagaaacggacgt ggacagatgc cgcgcactgc gcgaagaagt 1020 ggaaagctgg tacagagtggggattcagtg gccactgcag gtggctgccg ccacacgcta 1080 tggcgtgaac cacctgccgctggccacaat ggcgatggcc acgctcgccc aggacttgag 1140 atcgtacctg ggcgcgcgaggggagtacgt atccctctac gtctaacctt aatattttct 1200 gcatcatact tccaaacaatcatgtttccc atgcaattca ccaactcagc ctatcgccag 1260 atggagccca tgtttgcaccgggttcccga ggacaagtac agccgtaccg gccgcgcact 1320 aagcgccgcc aggagccgcaagtcggcaac gccgccatta ctgccctcgc gaaccagatg 1380 agtgcgctcc agttgcaggtagctggactt gccggccagg caagggtgga ccgccgtggg 1440 ccaagacgtg ttcagaagaacaagcagaag aagaagaact cttccaacgg agaaaaaccc 1500 aaagagaaga agaagaagcaaaaacaacag gagaagaagg gaagcggtgg cgaaaaagtc 1560 aagaagacta ggaaccgacccgggaaggag gtaaggatct ccgtaaagtg tgcccgacag 1620 agcaccttcc ccgtgtaccacgaaggtgct atatccggct acgctgtgct gattggatct 1680 cgcgtattca agccggcacacgtgaagggt aagatcgacc accctgaact ggcagacatc 1740 aagttccagg tcgccgaggacatggacctc gaagcagctg cgtacccgaa gagcatgcga 1800 gaccaagcgg ctgaaccagcgaccatgatg gacagagtgt acaactggga gtatggcact 1860 atcagagtgg aggataatgtcataatcgac gcaagcggta ggggcaagcc gggtgacagt 1920 ggcagggcca tcaccgacaactcgggaaag gttgttggta ttgtcctcgg aggaggaccc 1980 gatggcaggc gcacacgcctctccgtgata ggtttcgaca agaagatgaa ggctagggag 2040 atcgcctaca gtgatgccataccttggaca cgcgctccgg ccctcctgct gctgcctatg 2100 gttattgtct gcacctacaattccaacacc ttcgattgct ccaaaccgtc ctgccaggac 2160 tgctgcatta ctgctgaaccagagaaggcc atgaccatgc tgaaggacaa tctgaacgac 2220 ccgaactact gggacctactcattgctgtc accacctgtg gctccgcccg gagaaagagg 2280 gctgtgtcta cgtcgcctgccgccttttac gacacacaga tcctcgccgc ccacgcagct 2340 gcctccccat acagggcgtactgccccgat tgtgacggaa cagcgtgtat ctcgccgata 2400 gccatcgacg aggtggtgagcagtggcagc gaccacgtcc tccgcatgcg ggttggttct 2460 caatcgggag tgaccgctaagggtggtgcg gcgggtgaga cctctctgcg atacctggga 2520 agggacggga aggttcacgccgcagacaac acgcgactcg tggtgcgcac gactgcaaag 2580 tgcgacgtgc tgcaggccactggccactac atcctggcca actgcccagt ggggcagagc 2640 ctaaccgttg cggccacactggatggcacc cggcatcaat gcaccacggt tttcgaacac 2700 caagtaacgg agaagttcaccagagaacgc agcaagggcc accatctgtc cgacatgacc 2760 aagaaatgca ccagattttccactacacca aaaaagtccg ccctctacct cgttgatgtg 2820 tatgacgctc tgccgatttctgtagagatt agcaccgtcg taacatgcag cgacagccag 2880 tgcacagtga gggtgccacctggtaccaca gtgaaattcg acaagaaatg caagagcgct 2940 gactcggcaa ccgtcactttcaccagcgac tcccagacgt ttacgtgtga ggagccagtc 3000 ctaacggctg ccagtatcacccagggcaag ccacacctca gatcggcaat gttgcctagc 3060 ggaggcaagg aagtgaaagcaaggatcccg ttcccgttcc cgccggaaac cgcaacttgc 3120 agagtgagtg tagccccactgccgtcgatc acctacgagg aaagcgatgt cctgctagcc 3180 ggtaccgcaa aataccctgtgctgctaacc acacggaacc ttggtttcca tagcaacgcc 3240 acatccgaat ggatccagggcaagtacctg cgccgcatcc cggtcacgcc tcaagggatc 3300 gagctaacat ggggaaacaacgcgccgatg cacttttggt catccgtcag gtacgcatcc 3360 ggggacgctg atgcgtacccctgggaactt ctggtgtacc acaccaagca ccatccagag 3420 tacgcgtggg cgtttgtaggagttgcatgc ggcctgctgg ctatcgcagc gtgcatgttt 3480 gcgtgcgcat gcagcagggtgcggtactct ctggtcgcca acacgttcaa ctcgaaccca 3540 ccaccattga ccgcactgactgcagcactg tgttgcatac caggggctcg cgcggaccaa 3600 ccctacttgg acatcattgcctacttttta ggggtaagag ggtggtcagc cctgctggtc 3660 atccttgcgt atgtacagagctgcaagagc tacgaacaca ccgtggtggt cccaatggat 3720 ccaagagccc cgtcgtacgaagcagtgata aaccggaatg ggtatgatcc attgaagctg 3780 accatctcag tgaatttcaccgtcatctca ccaactacgg ctctggaata ttggacctgc 3840 gcaggagtcc ccatcgtcgagccgccccat gtgggctgct gcacgtcggt gtcctgcccc 3900 tctgacctct ctacgctgcatgcgtttact ggcaaagctg tctccgacgt gcactgcgat 3960 gtgcacacaa acgtgtaccccttgttgtgg ggcgcggctc actgcttctg ttccaccgag 4020 aatacacagg tcagcgctgtggcagccacc gtttctgagt tctgtgccca ggactcagag 4080 cgtgccgaag cgttcagcgtacacagcagc tcagtcaccg ctgaggtcct ggtgacgctt 4140 ggtgaagtgg tgacggcagtccacgtttac gtggacgggg taacatcagc caggggcact 4200 gacctcaaga tcgtggctggaccaataaca accgactact ccccattcga tcgcaaagta 4260 gtccgcatcg gcgaagaggtctataactat gactggcctc cttacggggc tggccgacca 4320 ggcacattcg gagacattcaagctaggtca accaactatg tcaaacccaa cgatctgtat 4380 ggggacatcg gaattgaagtactgcagccg actaacgacc acgtacatgt ggcttacacg 4440 tatacgacct ctgggttactgcgttggctg caggacgctc cgaaaccact cagtgtcaca 4500 gcaccgcacg gttgtaagatcagtgccaat ccgctcctgg ccctcgattg tggggttggt 4560 gccgtcccca tgtccatcaacattccggac gcgaagttta cccgcaaatt aaaggatccg 4620 aaaccatcgg ccctgaaatgcgtggtggac agctgcgagt acggggtgga ctacgggggc 4680 gccgccacga tcacctacgagggccacgag gccgggaagt gcgggattca ttccctgaca 4740 ccaggagtcc ccctgagaacatcggtggtt gaagtggttg ctggcgccaa taccgtcaaa 4800 acgaccttct cctcacccacgcccgaggtt gcactcgagg tagagatctg ttcggcaata 4860 gtgaagtgcg ctggtgagtgcactccaccg aaggaacatg tggtcgcaac caggcctcgc 4920 catggcagcg accctggaggctacatctcc gggcccgcaa tgcgctgggc cggagggatt 4980 gtagggaccc tagtggtcctgttccttatc cttgccgtca tctactgcgt ggtgaagaag 5040 tgccgctcca aaagaatccggatagtcaag agctaaattc cggtatacaa attgctcact 5100 aggagcccat ccgatcccacagggagtagg atgagtcatc tattggtttt aaaattttca 5160 atacaaaaaa aaaaaaaaa5179 2 394 PRT Salmon pancreatic disease virus NSP4 (C-terminal region)2 Thr Met Asp Ser Ala Ala Met Asn Val Glu Ala Phe Lys Ser Phe Ala 1 5 1015 Cys Lys Asp Thr Asp Leu Trp Thr Glu Phe Ala Glu Lys Pro Val Arg 20 2530 Leu Ser Pro Gly Gln Ile Glu Glu Tyr Val Phe His Leu Gln Gly Ala 35 4045 Lys Ala Asn Val Met His Ser Arg Val Glu Ala Val Cys Pro Asp Leu 50 5560 Ser Glu Val Ala Met Asp Arg Phe Thr Leu Asp Met Lys Arg Asp Val 65 7075 80 Lys Val Thr Pro Gly Thr Lys His Val Glu Glu Arg Pro Lys Val Gln 8590 95 Glu Ile Gln Ala Ala Asp Pro Met Ala Thr Ala Tyr Leu Cys Ala Ile100 105 110 His Arg Glu Leu Val Arg Arg Leu Lys Ala Val Leu Lys Pro SerIle 115 120 125 His Val Leu Phe Asp Met Ser Ser Glu Asp Phe Asp Ala IleVal Gly 130 135 140 His Gly Met Lys Leu Gly Asp Lys Val Leu Glu Thr AspIle Ser Ser 145 150 155 160 Phe Asp Lys Ser Gln Asp Gln Ala Met Ala ValThr Ala Leu Met Leu 165 170 175 Leu Arg Asp Leu Gly Val Glu Glu Asp LeuLeu Thr Leu Ile Glu Ala 180 185 190 Ser Phe Gly Asp Ile Thr Ser Ala HisLeu Pro Thr Gly Thr Arg Phe 195 200 205 Gln Phe Gly Ser Met Met Lys SerGly Leu Phe Leu Thr Leu Phe Val 210 215 220 Asn Thr Leu Leu Asn Ile ThrIle Ala Ala Arg Val Leu Arg Glu Gln 225 230 235 240 Leu Ala Asp Thr ArgCys Ala Ala Phe Ile Gly Asp Asp Asn Val Ile 245 250 255 Thr Gly Val ValSer Asp Asp Met Met Val Ala Arg Cys Ala Ser Trp 260 265 270 Leu Asn MetGlu Val Lys Ile Met Asp Met Glu Ile Gly Asn Met Ser 275 280 285 Pro TyrPhe Cys Gly Gly Phe Leu Leu Leu Asp Thr Val Thr Gly Thr 290 295 300 ValSer Arg Val Ser Asp Pro Val Lys Arg Leu Met Lys Met Gly Lys 305 310 315320 Pro Ala Leu Asn Asp Pro Glu Thr Asp Val Asp Arg Cys Arg Ala Leu 325330 335 Arg Glu Glu Val Glu Ser Trp Tyr Arg Val Gly Ile Gln Trp Pro Leu340 345 350 Gln Val Ala Ala Ala Thr Arg Tyr Gly Val Asn His Leu Pro LeuAla 355 360 365 Thr Met Ala Met Ala Thr Leu Ala Gln Asp Leu Arg Ser TyrLeu Gly 370 375 380 Ala Arg Gly Glu Tyr Val Ser Leu Tyr Val 385 390 31359 PRT Salmon pancreatic disease virus p130 3 Met Pro Arg Thr Ala ArgArg Ser Gly Lys Leu Val Gln Ser Gly Asp 1 5 10 15 Ser Val Ala Thr AlaGly Gly Cys Arg His Thr Leu Trp Arg Glu Pro 20 25 30 Pro Ala Ala Gly HisAsn Gly Asp Gly His Ala Arg Pro Gly Leu Glu 35 40 45 Ile Val Pro Gly ArgAla Arg Gly Val Arg Ile Pro Leu Arg Leu Thr 50 55 60 Leu Ile Phe Ser AlaSer Tyr Phe Gln Thr Ile Met Phe Pro Met Gln 65 70 75 80 Phe Thr Asn SerAla Tyr Arg Gln Met Glu Pro Met Phe Ala Pro Gly 85 90 95 Ser Arg Gly GlnVal Gln Pro Tyr Arg Pro Arg Thr Lys Arg Arg Gln 100 105 110 Glu Pro GlnVal Gly Asn Ala Ala Ile Thr Ala Leu Ala Asn Gln Met 115 120 125 Ser AlaLeu Gln Leu Gln Val Ala Gly Leu Ala Gly Gln Ala Arg Val 130 135 140 AspArg Arg Gly Pro Arg Arg Val Gln Lys Asn Lys Gln Lys Lys Lys 145 150 155160 Asn Ser Ser Asn Gly Glu Lys Pro Lys Glu Lys Lys Lys Lys Gln Lys 165170 175 Gln Gln Glu Lys Lys Gly Ser Gly Gly Glu Lys Val Lys Lys Thr Arg180 185 190 Asn Arg Pro Gly Lys Glu Val Arg Ile Ser Val Lys Cys Ala ArgGln 195 200 205 Ser Thr Phe Pro Val Tyr His Glu Gly Ala Ile Ser Gly TyrAla Val 210 215 220 Leu Ile Gly Ser Arg Val Phe Lys Pro Ala His Val LysGly Lys Ile 225 230 235 240 Asp His Pro Glu Leu Ala Asp Ile Lys Phe GlnVal Ala Glu Asp Met 245 250 255 Asp Leu Glu Ala Ala Ala Tyr Pro Lys SerMet Arg Asp Gln Ala Ala 260 265 270 Glu Pro Ala Thr Met Met Asp Arg ValTyr Asn Trp Glu Tyr Gly Thr 275 280 285 Ile Arg Val Glu Asp Asn Val IleIle Asp Ala Ser Gly Arg Gly Lys 290 295 300 Pro Gly Asp Ser Gly Arg AlaIle Thr Asp Asn Ser Gly Lys Val Val 305 310 315 320 Gly Ile Val Leu GlyGly Gly Pro Asp Gly Arg Arg Thr Arg Leu Ser 325 330 335 Val Ile Gly PheAsp Lys Lys Met Lys Ala Arg Glu Ile Ala Tyr Ser 340 345 350 Asp Ala IlePro Trp Thr Arg Ala Pro Ala Leu Leu Leu Leu Pro Met 355 360 365 Val IleVal Cys Thr Tyr Asn Ser Asn Thr Phe Asp Cys Ser Lys Pro 370 375 380 SerCys Gln Asp Cys Cys Ile Thr Ala Glu Pro Glu Lys Ala Met Thr 385 390 395400 Met Leu Lys Asp Asn Leu Asn Asp Pro Asn Tyr Trp Asp Leu Leu Ile 405410 415 Ala Val Thr Thr Cys Gly Ser Ala Arg Arg Lys Arg Ala Val Ser Thr420 425 430 Ser Pro Ala Ala Phe Tyr Asp Thr Gln Ile Leu Ala Ala His AlaAla 435 440 445 Ala Ser Pro Tyr Arg Ala Tyr Cys Pro Asp Cys Asp Gly ThrAla Cys 450 455 460 Ile Ser Pro Ile Ala Ile Asp Glu Val Val Ser Ser GlySer Asp His 465 470 475 480 Val Leu Arg Met Arg Val Gly Ser Gln Ser GlyVal Thr Ala Lys Gly 485 490 495 Gly Ala Ala Gly Glu Thr Ser Leu Arg TyrLeu Gly Arg Asp Gly Lys 500 505 510 Val His Ala Ala Asp Asn Thr Arg LeuVal Val Arg Thr Thr Ala Lys 515 520 525 Cys Asp Val Leu Gln Ala Thr GlyHis Tyr Ile Leu Ala Asn Cys Pro 530 535 540 Val Gly Gln Ser Leu Thr ValAla Ala Thr Leu Asp Gly Thr Arg His 545 550 555 560 Gln Cys Thr Thr ValPhe Glu His Gln Val Thr Glu Lys Phe Thr Arg 565 570 575 Glu Arg Ser LysGly His His Leu Ser Asp Met Thr Lys Lys Cys Thr 580 585 590 Arg Phe SerThr Thr Pro Lys Lys Ser Ala Leu Tyr Leu Val Asp Val 595 600 605 Tyr AspAla Leu Pro Ile Ser Val Glu Ile Ser Thr Val Val Thr Cys 610 615 620 SerAsp Ser Gln Cys Thr Val Arg Val Pro Pro Gly Thr Thr Val Lys 625 630 635640 Phe Asp Lys Lys Cys Lys Ser Ala Asp Ser Ala Thr Val Thr Phe Thr 645650 655 Ser Asp Ser Gln Thr Phe Thr Cys Glu Glu Pro Val Leu Thr Ala Ala660 665 670 Ser Ile Thr Gln Gly Lys Pro His Leu Arg Ser Ala Met Leu ProSer 675 680 685 Gly Gly Lys Glu Val Lys Ala Arg Ile Pro Phe Pro Phe ProPro Glu 690 695 700 Thr Ala Thr Cys Arg Val Ser Val Ala Pro Leu Pro SerIle Thr Tyr 705 710 715 720 Glu Glu Ser Asp Val Leu Leu Ala Gly Thr AlaLys Tyr Pro Val Leu 725 730 735 Leu Thr Thr Arg Asn Leu Gly Phe His SerAsn Ala Thr Ser Glu Trp 740 745 750 Ile Gln Gly Lys Tyr Leu Arg Arg IlePro Val Thr Pro Gln Gly Ile 755 760 765 Glu Leu Thr Trp Gly Asn Asn AlaPro Met His Phe Trp Ser Ser Val 770 775 780 Arg Tyr Ala Ser Gly Asp AlaAsp Ala Tyr Pro Trp Glu Leu Leu Val 785 790 795 800 Tyr His Thr Lys HisHis Pro Glu Tyr Ala Trp Ala Phe Val Gly Val 805 810 815 Ala Cys Gly LeuLeu Ala Ile Ala Ala Cys Met Phe Ala Cys Ala Cys 820 825 830 Ser Arg ValArg Tyr Ser Leu Val Ala Asn Thr Phe Asn Ser Asn Pro 835 840 845 Pro ProLeu Thr Ala Leu Thr Ala Ala Leu Cys Cys Ile Pro Gly Ala 850 855 860 ArgAla Asp Gln Pro Tyr Leu Asp Ile Ile Ala Tyr Phe Leu Gly Val 865 870 875880 Arg Gly Trp Ser Ala Leu Leu Val Ile Leu Ala Tyr Val Gln Ser Cys 885890 895 Lys Ser Tyr Glu His Thr Val Val Val Pro Met Asp Pro Arg Ala Pro900 905 910 Ser Tyr Glu Ala Val Ile Asn Arg Asn Gly Tyr Asp Pro Leu LysLeu 915 920 925 Thr Ile Ser Val Asn Phe Thr Val Ile Ser Pro Thr Thr AlaLeu Glu 930 935 940 Tyr Trp Thr Cys Ala Gly Val Pro Ile Val Glu Pro ProHis Val Gly 945 950 955 960 Cys Cys Thr Ser Val Ser Cys Pro Ser Asp LeuSer Thr Leu His Ala 965 970 975 Phe Thr Gly Lys Ala Val Ser Asp Val HisCys Asp Val His Thr Asn 980 985 990 Val Tyr Pro Leu Leu Trp Gly Ala AlaHis Cys Phe Cys Ser Thr Glu 995 1000 1005 Asn Thr Gln Val Ser Ala ValAla Ala Thr Val Ser Glu Phe Cys Ala 1010 1015 1020 Gln Asp Ser Glu ArgAla Glu Ala Phe Ser Val His Ser Ser Ser Val 1025 1030 1035 1040 Thr AlaGlu Val Leu Val Thr Leu Gly Glu Val Val Thr Ala Val His 1045 1050 1055Val Tyr Val Asp Gly Val Thr Ser Ala Arg Gly Thr Asp Leu Lys Ile 10601065 1070 Val Ala Gly Pro Ile Thr Thr Asp Tyr Ser Pro Phe Asp Arg LysVal 1075 1080 1085 Val Arg Ile Gly Glu Glu Val Tyr Asn Tyr Asp Trp ProPro Tyr Gly 1090 1095 1100 Ala Gly Arg Pro Gly Thr Phe Gly Asp Ile GlnAla Arg Ser Thr Asn 1105 1110 1115 1120 Tyr Val Lys Pro Asn Asp Leu TyrGly Asp Ile Gly Ile Glu Val Leu 1125 1130 1135 Gln Pro Thr Asn Asp HisVal His Val Ala Tyr Thr Tyr Thr Thr Ser 1140 1145 1150 Gly Leu Leu ArgTrp Leu Gln Asp Ala Pro Lys Pro Leu Ser Val Thr 1155 1160 1165 Ala ProHis Gly Cys Lys Ile Ser Ala Asn Pro Leu Leu Ala Leu Asp 1170 1175 1180Cys Gly Val Gly Ala Val Pro Met Ser Ile Asn Ile Pro Asp Ala Lys 11851190 1195 1200 Phe Thr Arg Lys Leu Lys Asp Pro Lys Pro Ser Ala Leu LysCys Val 1205 1210 1215 Val Asp Ser Cys Glu Tyr Gly Val Asp Tyr Gly GlyAla Ala Thr Ile 1220 1225 1230 Thr Tyr Glu Gly His Glu Ala Gly Lys CysGly Ile His Ser Leu Thr 1235 1240 1245 Pro Gly Val Pro Leu Arg Thr SerVal Val Glu Val Val Ala Gly Ala 1250 1255 1260 Asn Thr Val Lys Thr ThrPhe Ser Ser Pro Thr Pro Glu Val Ala Leu 1265 1270 1275 1280 Glu Val GluIle Cys Ser Ala Ile Val Lys Cys Ala Gly Glu Cys Thr 1285 1290 1295 ProPro Lys Glu His Val Val Ala Thr Arg Pro Arg His Gly Ser Asp 1300 13051310 Pro Gly Gly Tyr Ile Ser Gly Pro Ala Met Arg Trp Ala Gly Gly Ile1315 1320 1325 Val Gly Thr Leu Val Val Leu Phe Leu Ile Leu Ala Val IleTyr Cys 1330 1335 1340 Val Val Lys Lys Cys Arg Ser Lys Arg Ile Arg IleVal Lys Ser 1345 1350 1355 4 282 PRT Salmon pancreatic disease viruscapsid 4 Met Phe Pro Met Gln Phe Thr Asn Ser Ala Tyr Arg Gln Met Glu Pro1 5 10 15 Met Phe Ala Pro Gly Ser Arg Gly Gln Val Gln Pro Tyr Arg ProArg 20 25 30 Thr Lys Arg Arg Gln Glu Pro Gln Val Gly Asn Ala Ala Ile ThrAla 35 40 45 Leu Ala Asn Gln Met Ser Ala Leu Gln Leu Gln Val Ala Gly LeuAla 50 55 60 Gly Gln Ala Arg Val Asp Arg Arg Gly Pro Arg Arg Val Gln LysAsn 65 70 75 80 Lys Gln Lys Lys Lys Asn Ser Ser Asn Gly Glu Lys Pro LysGlu Lys 85 90 95 Lys Lys Lys Gln Lys Gln Gln Glu Lys Lys Gly Ser Gly GlyGlu Lys 100 105 110 Val Lys Lys Thr Arg Asn Arg Pro Gly Lys Glu Val ArgIle Ser Val 115 120 125 Lys Cys Ala Arg Gln Ser Thr Phe Pro Val Tyr HisGlu Gly Ala Ile 130 135 140 Ser Gly Tyr Ala Val Leu Ile Gly Ser Arg ValPhe Lys Pro Ala His 145 150 155 160 Val Lys Gly Lys Ile Asp His Pro GluLeu Ala Asp Ile Lys Phe Gln 165 170 175 Val Ala Glu Asp Met Asp Leu GluAla Ala Ala Tyr Pro Lys Ser Met 180 185 190 Arg Asp Gln Ala Ala Glu ProAla Thr Met Met Asp Arg Val Tyr Asn 195 200 205 Trp Glu Tyr Gly Thr IleArg Val Glu Asp Asn Val Ile Ile Asp Ala 210 215 220 Ser Gly Arg Gly LysPro Gly Asp Ser Gly Arg Ala Ile Thr Asp Asn 225 230 235 240 Ser Gly LysVal Val Gly Ile Val Leu Gly Gly Gly Pro Asp Gly Arg 245 250 255 Arg ThrArg Leu Ser Val Ile Gly Phe Asp Lys Lys Met Lys Ala Arg 260 265 270 GluIle Ala Tyr Ser Asp Ala Ile Pro Trp 275 280 5 71 PRT Salmon pancreaticdisease virus E3 5 Thr Arg Ala Pro Ala Leu Leu Leu Leu Pro Met Val IleVal Cys Thr 1 5 10 15 Tyr Asn Ser Asn Thr Phe Asp Cys Ser Lys Pro SerCys Gln Asp Cys 20 25 30 Cys Ile Thr Ala Glu Pro Glu Lys Ala Met Thr MetLeu Lys Asp Asn 35 40 45 Leu Asn Asp Pro Asn Tyr Trp Asp Leu Leu Ile AlaVal Thr Thr Cys 50 55 60 Gly Ser Ala Arg Arg Lys Arg 65 70 6 438 PRTSalmon pancreatic disease virus E2 6 Ala Val Ser Thr Ser Pro Ala Ala PheTyr Asp Thr Gln Ile Leu Ala 1 5 10 15 Ala His Ala Ala Ala Ser Pro TyrArg Ala Tyr Cys Pro Asp Cys Asp 20 25 30 Gly Thr Ala Cys Ile Ser Pro IleAla Ile Asp Glu Val Val Ser Ser 35 40 45 Gly Ser Asp His Val Leu Arg MetArg Val Gly Ser Gln Ser Gly Val 50 55 60 Thr Ala Lys Gly Gly Ala Ala GlyGlu Thr Ser Leu Arg Tyr Leu Gly 65 70 75 80 Arg Asp Gly Lys Val His AlaAla Asp Asn Thr Arg Leu Val Val Arg 85 90 95 Thr Thr Ala Lys Cys Asp ValLeu Gln Ala Thr Gly His Tyr Ile Leu 100 105 110 Ala Asn Cys Pro Val GlyGln Ser Leu Thr Val Ala Ala Thr Leu Asp 115 120 125 Gly Thr Arg His GlnCys Thr Thr Val Phe Glu His Gln Val Thr Glu 130 135 140 Lys Phe Thr ArgGlu Arg Ser Lys Gly His His Leu Ser Asp Met Thr 145 150 155 160 Lys LysCys Thr Arg Phe Ser Thr Thr Pro Lys Lys Ser Ala Leu Tyr 165 170 175 LeuVal Asp Val Tyr Asp Ala Leu Pro Ile Ser Val Glu Ile Ser Thr 180 185 190Val Val Thr Cys Ser Asp Ser Gln Cys Thr Val Arg Val Pro Pro Gly 195 200205 Thr Thr Val Lys Phe Asp Lys Lys Cys Lys Ser Ala Asp Ser Ala Thr 210215 220 Val Thr Phe Thr Ser Asp Ser Gln Thr Phe Thr Cys Glu Glu Pro Val225 230 235 240 Leu Thr Ala Ala Ser Ile Thr Gln Gly Lys Pro His Leu ArgSer Ala 245 250 255 Met Leu Pro Ser Gly Gly Lys Glu Val Lys Ala Arg IlePro Phe Pro 260 265 270 Phe Pro Pro Glu Thr Ala Thr Cys Arg Val Ser ValAla Pro Leu Pro 275 280 285 Ser Ile Thr Tyr Glu Glu Ser Asp Val Leu LeuAla Gly Thr Ala Lys 290 295 300 Tyr Pro Val Leu Leu Thr Thr Arg Asn LeuGly Phe His Ser Asn Ala 305 310 315 320 Thr Ser Glu Trp Ile Gln Gly LysTyr Leu Arg Arg Ile Pro Val Thr 325 330 335 Pro Gln Gly Ile Glu Leu ThrTrp Gly Asn Asn Ala Pro Met His Phe 340 345 350 Trp Ser Ser Val Arg TyrAla Ser Gly Asp Ala Asp Ala Tyr Pro Trp 355 360 365 Glu Leu Leu Val TyrHis Thr Lys His His Pro Glu Tyr Ala Trp Ala 370 375 380 Phe Val Gly ValAla Cys Gly Leu Leu Ala Ile Ala Ala Cys Met Phe 385 390 395 400 Ala CysAla Cys Ser Arg Val Arg Tyr Ser Leu Val Ala Asn Thr Phe 405 410 415 AsnSer Asn Pro Pro Pro Leu Thr Ala Leu Thr Ala Ala Leu Cys Cys 420 425 430Ile Pro Gly Ala Arg Ala 435 7 32 PRT Salmon pancreatic disease virus 6K7 Asp Gln Pro Tyr Leu Asp Ile Ile Ala Tyr Phe Leu Gly Val Arg Gly 1 5 1015 Trp Ser Ala Leu Leu Val Ile Leu Ala Tyr Val Gln Ser Cys Lys Ser 20 2530 8 461 PRT Salmon pancreatic disease virus E1 8 Tyr Glu His Thr ValVal Val Pro Met Asp Pro Arg Ala Pro Ser Tyr 1 5 10 15 Glu Ala Val IleAsn Arg Asn Gly Tyr Asp Pro Leu Lys Leu Thr Ile 20 25 30 Ser Val Asn PheThr Val Ile Ser Pro Thr Thr Ala Leu Glu Tyr Trp 35 40 45 Thr Cys Ala GlyVal Pro Ile Val Glu Pro Pro His Val Gly Cys Cys 50 55 60 Thr Ser Val SerCys Pro Ser Asp Leu Ser Thr Leu His Ala Phe Thr 65 70 75 80 Gly Lys AlaVal Ser Asp Val His Cys Asp Val His Thr Asn Val Tyr 85 90 95 Pro Leu LeuTrp Gly Ala Ala His Cys Phe Cys Ser Thr Glu Asn Thr 100 105 110 Gln ValSer Ala Val Ala Ala Thr Val Ser Glu Phe Cys Ala Gln Asp 115 120 125 SerGlu Arg Ala Glu Ala Phe Ser Val His Ser Ser Ser Val Thr Ala 130 135 140Glu Val Leu Val Thr Leu Gly Glu Val Val Thr Ala Val His Val Tyr 145 150155 160 Val Asp Gly Val Thr Ser Ala Arg Gly Thr Asp Leu Lys Ile Val Ala165 170 175 Gly Pro Ile Thr Thr Asp Tyr Ser Pro Phe Asp Arg Lys Val ValArg 180 185 190 Ile Gly Glu Glu Val Tyr Asn Tyr Asp Trp Pro Pro Tyr GlyAla Gly 195 200 205 Arg Pro Gly Thr Phe Gly Asp Ile Gln Ala Arg Ser ThrAsn Tyr Val 210 215 220 Lys Pro Asn Asp Leu Tyr Gly Asp Ile Gly Ile GluVal Leu Gln Pro 225 230 235 240 Thr Asn Asp His Val His Val Ala Tyr ThrTyr Thr Thr Ser Gly Leu 245 250 255 Leu Arg Trp Leu Gln Asp Ala Pro LysPro Leu Ser Val Thr Ala Pro 260 265 270 His Gly Cys Lys Ile Ser Ala AsnPro Leu Leu Ala Leu Asp Cys Gly 275 280 285 Val Gly Ala Val Pro Met SerIle Asn Ile Pro Asp Ala Lys Phe Thr 290 295 300 Arg Lys Leu Lys Asp ProLys Pro Ser Ala Leu Lys Cys Val Val Asp 305 310 315 320 Ser Cys Glu TyrGly Val Asp Tyr Gly Gly Ala Ala Thr Ile Thr Tyr 325 330 335 Glu Gly HisGlu Ala Gly Lys Cys Gly Ile His Ser Leu Thr Pro Gly 340 345 350 Val ProLeu Arg Thr Ser Val Val Glu Val Val Ala Gly Ala Asn Thr 355 360 365 ValLys Thr Thr Phe Ser Ser Pro Thr Pro Glu Val Ala Leu Glu Val 370 375 380Glu Ile Cys Ser Ala Ile Val Lys Cys Ala Gly Glu Cys Thr Pro Pro 385 390395 400 Lys Glu His Val Val Ala Thr Arg Pro Arg His Gly Ser Asp Pro Gly405 410 415 Gly Tyr Ile Ser Gly Pro Ala Met Arg Trp Ala Gly Gly Ile ValGly 420 425 430 Thr Leu Val Val Leu Phe Leu Ile Leu Ala Val Ile Tyr CysVal Val 435 440 445 Lys Lys Cys Arg Ser Lys Arg Ile Arg Ile Val Lys Ser450 455 460 9 37 DNA Artificial Sequence Description of ArtificialSequence primer 9 tgcatgcggc cgcatgtttc ccatgcaatt caccaac 37 10 37 DNAArtificial Sequence Description of Artificial Sequence primer 10tgcatgcggc cgcttgtatt gaaaatttta aaaccaa 37 11 37 DNA ArtificialSequence Description of Artificial Sequence primer 11 tgcatgcggccgcatgacac gcgctccggc cctcctg 37 12 40 DNA Artificial SequenceDescription of Artificial Sequence primer 12 tgcatgcggc cgctcacgcgcgagcccctg gtatgcaaca 40 13 37 DNA Artificial Sequence Description ofArtificial Sequence primer 13 tgcatgcggc cgcatggctg tgtctacgtc gcctgcc37 14 204 DNA Salmon pancreatic disease virus CDS (1)..(204) 6K 14 gaccaa ccc tac ttg gac atc att gcc tac ttg tgg acc aac agc aaa 48 Asp GlnPro Tyr Leu Asp Ile Ile Ala Tyr Leu Trp Thr Asn Ser Lys 1 5 10 15 gtggcc ttc ggg cta caa ttt gcg gcg ccc gtg gcc tgt gtg ctc atc 96 Val AlaPhe Gly Leu Gln Phe Ala Ala Pro Val Ala Cys Val Leu Ile 20 25 30 att acatac gcc ctt agg cac tgc aga ttg tgc tgc aag tct ttt tta 144 Ile Thr TyrAla Leu Arg His Cys Arg Leu Cys Cys Lys Ser Phe Leu 35 40 45 ggg gta agaggg tgg tca gcc ctg ctg gtc atc ctt gcg tat gta cag 192 Gly Val Arg GlyTrp Ser Ala Leu Leu Val Ile Leu Ala Tyr Val Gln 50 55 60 agc tgc aag agc204 Ser Cys Lys Ser 65 15 68 PRT Salmon pancreatic disease virus 15 AspGln Pro Tyr Leu Asp Ile Ile Ala Tyr Leu Trp Thr Asn Ser Lys 1 5 10 15Val Ala Phe Gly Leu Gln Phe Ala Ala Pro Val Ala Cys Val Leu Ile 20 25 30Ile Thr Tyr Ala Leu Arg His Cys Arg Leu Cys Cys Lys Ser Phe Leu 35 40 45Gly Val Arg Gly Trp Ser Ala Leu Leu Val Ile Leu Ala Tyr Val Gln 50 55 60Ser Cys Lys Ser 65

1. A structural protein of Fish Pancreatic Disease virus, wherein theprotein is selected from the group consisting of a capsid protein, an E1protein, an E2 protein, an E3 protein and a 6K protein.
 2. Thestructural protein of claim 1, wherein the protein comprises an aminoacid sequence selected from the group consisting of SEQ ID NO:3, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:15.
 3. Anucleic acid comprising a nucleotide sequence encoding one or more ofthe structural proteins of claim
 1. 4. The nucleic acid of claim 3,wherein the nucleotide sequence comprises one or more of SEQ ID NO 1 andSEQ ID NO 14, or fragments thereof.
 5. A pharmaceutical composition,comprising: a protein of claim 1 and a pharmaceutically acceptablecarrier.
 6. (Canceled).
 7. A DNA vaccine, comprising: a pharmaceuticallyacceptable carrier and a DNA plasmid in which a nucleotide sequence ofclaim 3 is operably linked to a transcriptional regulatory sequence. 8.A vector vaccine, comprising: a live attenuated modified bacteria orvirus, wherein the bacteria or virus comprises one or more of thenucleotide sequences of claim
 3. 9. A vaccine, comprising: one or moreof the structural PD proteins of claim 1 and a pharmaceuticallyacceptable carrier.
 10. A diagnostic kit, comprising: one or moreproteins of claim
 1. 11. A pharmaceutical composition, comprising: anucleic acid of claim 1 and a pharmaceutically acceptable carrier.
 12. Adiagnostic kit, comprising: one or more nucleic acids, or fragmentsthereof, of claim 3.